Establishment of a DAS-ELISA detection method for thermostable direct hemolysin of Vibrio parahaemolyticus
BAI Xue-xin1,2, HU Chen-yi2, JIN Zhi-ying2, WAN Wei2, LI Yue2, WANG Jing2, LI Yan-wei2, GAO Shan2, WANG Jing-lin1,2
1. School of Basic Medical Sciences, Anhui Medical University, Hefei 230032,China; 2. State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China
Abstract：This study aimed to establish a double antibody sandwich ELISA (DAS-ELISA) for the detection of thermostable direct hemolysin in Vibrio parahaemolyticus. The gene sequence of thermostable direct hemolysin of Vibrio parahaemolyticus was amplified by PCR and cloned into the pET-28a vector for prokaryotic expression, and the expressed protein was purified and identified. New Zealand White rabbits were immunized with the high purity recombinant protein to prepare polyclonal antibodies. The affinity of the antibodies was determined on the basis of molecular interactions to develop a DAS-ELISA method for the detection of thermostable direct hemolysin in Vibrio parahaemolyticus. The reaction conditions were optimized through the tessellation method, a standard curve was established, and the performance of the established DAS-ELISA method was evaluated. A polyclonal antibody with an affinity of 1×10-8 was prepared, and the sensitivity of the DAS-ELISA method with this high affinity antibody was 78 ng/mL, with a quantitative range of 78-312 ng/mL. No cross-reactivity with Vibrio vulnificus hemolysin, Clostridium perfringens alpha toxin and Clostridium perfringens epsilon toxin was observed. The sensitivity of the method was not altered in the detection in complex matrices, evaluated by addition of recombinant proteins to the standard curves of the three mock samples prepared from scallops, clams and arks. The detection rate was 100% when the method was evaluated by addition of bacterial supernatant to the three mock samples. Thus, a DAS-ELISA method for the detection of thermostable direct hemolysin of V. parahaemolyticus was successfully constructed. This method is specific, sensitive and suitable for the detection of complex matrices. It is expected to be developed into a rapid diagnostic kit for thermostable direct hemolysin of V. thus indicating its good application prospects.
白雪欣, 胡宸艺, 金志颖, 万伟, 李月, 王菁, 李岩伟, 高姗, 王景林. 副溶血弧菌耐热直接溶血素DAS-ELISA检测方法的建立[J]. 中国人兽共患病学报, 2022, 38(8): 666-672.
BAI Xue-xin, HU Chen-yi, JIN Zhi-ying, WAN Wei, LI Yue, WANG Jing, LI Yan-wei, GAO Shan, WANG Jing-lin. Establishment of a DAS-ELISA detection method for thermostable direct hemolysin of Vibrio parahaemolyticus. Chinese Journal of Zoonoses, 2022, 38(8): 666-672.
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2022, Vol. 38 
